ABSTRACT: Acute infection with malaria is often accompanied by the symptoms of a systemic cytokine storm. These symptoms result from a systemic inflammatory response via the activation of phagocytes. The overall goal of our ongoing project is to define the mechanisms of innate immune activation and regulation that account for the clinical symptoms and complications of malaria. This grant is a renewal of R01 AI079293. This RO1 has been highly productive having resulted in 40 publications including 16 peer-reviewed manuscripts of original work in the latest funding period (9 of these directly related to the grant, an additional 5 papers were supported by grant-related infrastructure in Brazil). Over the last decade, we have defined what we believe to be the principal ?malaria toxins? and their cognate receptors: parasite AT-rich DNA, immune complexes, hemozoin, free heme, and GPI anchors. We found that these ligands drive Toll-like receptors (TLRs 2, 3, 4, 9), NOD-like receptors (NLRP3, NLRP12), absent in melanoma 2 (AIM2), and the cGAS-STING pathway. Our work led to recognition that a cytosolic DNA binding protein Cnbp, regulates IL12b and IFNg and, under certain conditions, type I interferons. We have described the phenomena of innate immune memory in malaria, which is one part of an epigenetic process that may both protect the human host or, under certain conditions, might result in severe malaria (especially if accompanied by bacterial infection). More recently, we have identified several lncRNAs in malaria-stimulated phagocytes, many of which might regulate clinical disease. Thus, we have accomplished most of what we set out to do 5 years ago. In this renewal, we plan to focus on identifying the most basic methods by which the innate immune system is regulated in malaria by combining cell culture studies, in vitro mouse work with the analysis of patient samples. There are three Aims. The major hypothesis of the grant is that the innate immune response is highly regulated via epigenetic modifications of chromatin as well as at the transcriptional level. This regulation involves the interaction of lymphoid cells with phagocytes, resulting in innate immune training and a variety of epigenetic changes. The epigenetic changes involve the modification of chromatin, alterations of chromatin accessibility (Aim 1). In addition, inflammatory signals during malaria result in the generation of long non-coding RNAs including LUCAT-1 and LIMIT, whose roles we will explore in detail in vitro, in a LUCAT-1 KO mouse and in human clinical samples (Aim 2). Finally, we will define the role of a transcriptional regulator known as Cnbp, a nucleic acid binding protein that we initially thought to be a DNA sensor. Cnbp has proven to be involved in regulating IL12beta transcription and expression via its interaction with c-Rel and the IL12 promoter (Aim 3). In all of the Aims described briefly above, we will analyze leukocytes from febrile patients with malaria (both P. vivax and P falciparum) from our site in Porto Velho, Brazil. This grant is an ambitious project built upon the successful completion of two previously ambitious grants. Over the next funding cycle, we hope to contribute significantly more to our knowledge of innate immunity in malaria.